Antibodies and specific alternatives are a standard tool for research product, diagnostic, and therapeutic applications. Discovery and characterization of affinity reagents for these applications can be challenging and arduous, involving antigen preparation, in vitro and/or in vivo development of binders, as well as screening and isolation of those binders. For example, mouse monoclonal antibodies are generated by immunization of mice with a purified antigen, to allow in vivo development of IgG antibodies by the B cells, and selection of an appropriate antibody by screening the expression of hybridomas (Köhler & Milstein Nature (1975) 256(5517):495-7). More recently, antibody fragments (e.g., single chain variable fragments (scFv), and VHH domains) and artificial affinity binders (e.g., Affibodies, Monobodies, and DARPins) have been created and are developed by screening large gene libraries of potential binders with various panning technologies. These technologies have allowed the development of numerous protein scaffolds with unique affinity interaction domains that bind target epitopes.
A plethora of affinity molecule panning/screening technologies have been developed over the past decade and all share the requisite association of expressed protein with its nucleic acid coding sequence, which serves to identify the affinity binder. These technologies can be generally divided into two groups: in vivo and in vitro display. In vivo technologies are based on the introduction by viral infection or cellular transfection of a single gene into a cell, expression of the affinity binder protein from the gene, delivery of the binder to the surface of the cell or phage, selection of the affinity molecule to an immobilized target molecule, and identification of the gene associated with the affinity binder (Hoogenboom, H. R. (2005) Nature Biotech 23(9), 1105-16). Examples of in vivo type display technologies are bacterial, yeast, mammalian, insect, and phage display.
In vitro technologies use the basic protein expression apparatus of a cell, either as a cell extract or a purified system (Shimizu, et al. Nature Biotechnology (2001) 19, 751-755), but do not require a viable cell to express the affinity binder. Therefore, the required association of coding sequence with affinity binder is through a physical bond. For ribosome display, this is done by “freezing” the ribosome at the end (stop codon) of an mRNA transcript after it has completed translating the transcript into a protein, which is also bound to the ribosome (Hanes, J. et al. (1998) Proc Natl Acad Sci USA 95(24), 14130-5). Affinity molecules to the target molecule are selected similarly to in vivo display technologies (i.e., with an immobilized target molecule) and the mRNA transcript reverse transcribed into DNA for amplification, identification, and cloning. RNA display covalently links the 3′ end of an mRNA transcript to the translated affinity molecule protein using a linker, which is added to the 3′ ends of the mRNA and incorporated into the affinity binder protein at its C-terminal end (Roberts, R. W., and Szostak, J. W. (1997) PNAS 94(23), 12297-302). DNA display physically associates the affinity molecule to the DNA coding sequence, either using a DNA replication initiator protein (RepA) fused to the affinity binder (ref) or a Hae III DNA methyltransferase that specifically recognizes methylated sequences (Bertschinger & Neri Protein (2004) Eng. Des. Sel. 17(9), 699-707). While the former can be performed in solution, the latter requires individual reactions for each protein expression event using in vitro compartmentalization.
In vitro compartmentalization (IVC) was developed in 1998 by Andrew Griffiths and Dan Tawfik (Nature Biotech. 16, 652) as an alternative to standard reaction vessels. Using cellular compartmentalization as a model, this technology facilitates the creation of minuscule aqueous solutions using water-in-oil emulsions, that is, small droplets of hydrophilic fluid exist as individual compartments in a sea of hydrophobic fluid. Droplets can be less than a micron in size (less than a femtoliter in volume) and an emulsion can have greater than 1010 droplets per ml. Griffiths and Tawfik demonstrated that a gene library distributed in a cell-free extract and compartmentalized into droplets can express their individual proteins in each droplet. In one case, the protein is an enzyme that reacts with a substrate and the technique can be used to evolve the enzyme with desired attributes. In another case, the gene is covalently bound to a bead that also contains an affinity molecule that captures the gene product (e.g., using a protein tag), thereby associating the gene with its expression product for affinity molecule selection. In addition, there is the technique noted above that uses Hae III DNA methyltransferase.
Homogeneous noncompetitive immunoassays by definition do not require physical separation of an affinity molecule bound to its target before detection. A common example of this technique is aggregation or agglutination immunoassays. Another example is Förster (or fluorescence) resonance energy transfer (FRET), which is based on the transfer of Förster energy (nonradiative transfer) from an excited fluorophore to another fluorophore that is in proximity (Valanne et al. (2005) Anal. Chim. Acta 539, 251-6). A similar method uses a bioluminescent protein, such as luciferase, to excite a proximal fluorophore (BRET), typically a fluorescent protein (Xu et al. (1999) Proc. Natl. Acad. Sci. USA 96(1), 151-6). Another homogeneous assay alternative is a luminescent oxygen-channeling chemistry (Ullman et al. (1994) Proc. Natl. Acad. Sci. USA 91(12), 5426-30), wherein a light induced singlet oxygen generating system transfers the singlet oxygen to a chemiluminescent system in proximity. The NanoDLSay system is a single-step homogeneous assay that uses conjugated gold particles to form aggregates in the presence of an antigen (Liu et al (2008) J. Am. Chem. Soc. 130 (9), 2780-2). Proximity ligation assay (PLA) uses two DNA single strands, one attached to each affinity molecule partner, that are complementary to a third oligonucleotide (Gullberg (2004) Proc Natl Acad Sci USA 101(22), 8420-8424). When the affinity molecules are proximal to each other, the strands hybridize to the linker oligonucleotide in an orientation where ends (3′ and 5′) are next to each other and can be ligated together. The resulting DNA is amplified and quantified using Q-PCR.
Protein fragment compartmentalization (PFC) is similar to PLA in that 2 complementary molecules are fused to potentially proximal binders that interact preferentially when in proximity. In this case, the molecules are protein fragments capable of assembling into a complete and functional protein, typically an enzyme or fluorescent protein. Protein-protein interaction sensors using protein fragments were first developed by Nils Johnsson and Alexander Varshaysky using a split ubiquitin and this idea was further developed by Stephen Michnik in 1997 (Pelletier et al. (1998) J. Biomol. Tech. acc. No. 50012) as an in vivo protein-protein interaction analysis tool. The technique was used to develop an in vivo antibody (scFv) screening method by fusing one protein fragment on the antigen and the other protein fragment on a library of scFv (Mössner et al. J. Mol. Biol. (2001) 308(2), 115-122; Koch et al. J. Mol. Biol. (2006) 357, 427-441; Secco et al. (2009) Prot. Evol. Des. Sel. 22(5), 149-158). Recently, Panbio Diagnostics has developed a homogeneous assay for the detection of antigen or antibodies using protein fragment complementation, which they call Forced Enzyme Complementation (FEC).
Most examples of affinity binder screening by PFC are in vivo, that is, the binding reactions are compartmentalized using cells. As mentioned above, an alternative to using live cells is encapsulated cell-free extracts using IVC, preferably manipulated using microfluidics. While there are numerous examples of in vitro protein expression using IVC, only recently has this been done using microfluidic devices. Dittrich, et al. (Chembiochem. (2005) 6(5):811-4), has recently demonstrated in vitro expression of a green fluorescent protein (red-shifted mutant) in 5 micron (˜65 fL) microdroplets that were detected using confocal fluoroscopy. Few other researchers have developed this technology, preferring to use compartmentalized cell based assays (Brouzes et al. PNAS (2009) early edition).